1.1 Field of Invention
The present invention relates generally to the field of molecular biology. More particularly, certain embodiments concern methods and compositions for the bacterial production of heterologous disulfide bond-containing polypeptides. In a preferred embodiment the eukaryotic protein, tissue plasminogen activator (tPA), and variants thereof, are produced in E. coli cells using recombinant vectors which direct the coexpression of these proteins with a prokaryotic enzyme, such as DsbC or DsbG.
1.2 Description of the Related Art
1.2.1 Protein Expression in Bacterial Hosts
A significant achievement in molecular biology has been the use of recombinant bacterial cells to produce eukaryotic proteins. This method has been particularly useful for production of medically important polypeptides that are obtained in low yield from natural sources. Often otherwise difficult to obtain in quantity, such proteins are "overexpressed" in the host cell and subsequently isolated and purified. Preinsulin for example may be produced in a recombinant prokaryotic microorganism carrying DNA encoding rat preinsulin (U.S. Pat. Nos. 4,431,740 and 4,652,525, each specifically incorporated herein by reference).
Expression of multiple disulfide bond-containing eukaryotic polypeptides, and particularly mammalian proteins, in bacterial cells has frequently produced disappointing and unsatisfactory results because conditions and environment in the host cells were not conducive to correct folding. Disulfide bond formation is a process mainly restricted to proteins outside the cytoplasmic compartment such as those secreted into the lumen of the endoplasmic reticulum (ER) or the periplasm of gram negative bacteria. Correct folding may depend on the formation of cysteine-cysteine linkages and subsequent stabilization of the protein into an enzymatically active structure. However, the cytoplasm is in fact a reducing environment due to the presence of thioredoxin reductase or reduced glutathione, thus blocking oxidation so that disulfide bonds do not form. The endoplasmic reticulum (ER) apparently is more conducive to oxidation due to the presence of oxygen or oxidized glutathione.
Recent studies indicate that disulfide bond formation in vivo is a catalyzed process, either in the ER or periplasm. In E. coli, a pathway for the formation of disulfide bonds in secreted proteins has been described, involving two proteins, DsbA and DsbB (Bardwell et al., 1993a; 1993b; Missiakas et al., 1993).
A role for these Dsb proteins is supported by the observation that mutants of E. coli that lack DsbA or DsbB are defective with respect to disulfide bond formation (Dailey and Berg, 1993). In the yeast Saccharomyces cerevisiae, a similar defect is found in certain mutants defective in protein disulfide isomerase (PDI) gene. Disulfide bond formation in carboxypeptidase Y in these mutants is impaired.
1.2.2 Recombinant Expression of Eukaryotic Proteins in Bacterial Hosts
It is known that disulfide bonds are critical in some proteins in order for proper folding and even in transport and secretion. Yet many proteins cannot be efficiently expressed in bacterial hosts due to failure of disulfide bond formation. Cytoplasmic expression systems in bacteria are not conducive to disulfide bond formation because of a reducing environment. The presence of proteases in the cytoplasm may cause rapid degradation of the protein, resulting in low yields.
1.2.3 Protein Folding in Vitro and in Vivo
Much research has been conducted in the field of protein folding showing that, in vitro, reduced and denatured ribonuclease could refold into the active enzyme with the formation of suitable disulfide bonds. Later, a catalyst responsible for oxidative folding in eukaryotes was discovered, called protein disulfide isomerase (PDI).
Two types of proteins that assist in protein folding have been described: non-catalytic molecular chaperones that presumably prevent improper interactions leading to aggregation and events other than proper folding, and catalysts for two steps in protein folding, cis-trans prolyl isomerization and disulfide bond formation. While clear evidence for an in vivo requirement of prolyl isomerase activity is still lacking, the relatively recent isolation of mutants that are severely defective in disulfide bond formation has confirmed that this latter folding step is catalyzed in vivo.
U.S. Pat. Nos. 5,270,181 and 5,292,646 (specifically incorporated herein by reference in their entirety) disclose recombinant production of heterologous proteins by expression as a fusion protein with a thioredoxin-like protein (such as the thioredoxin-like domain of PDI) for high stability and solubility. Jap. Pat. Appl. No. JP 60-38771 discloses the expression of a human PDI gene linked to human serum albumin pre-pro sequence and co-expression of this linked gene and a foreign gene encoding a polypeptide. Intl. Pat. Appl. Publ. No. WO 93/25676 discloses the production of disulfide-bonded recombinant proteins using a PDI, preferably a yeast PDI. Eur. Pat. Appl. No. EP 293,793 discloses a polypeptide with PDI activity ensuring natural disulfide bridge arrangement in recombinant proteins. Intl. Pat. Appl. Publ. No. WO 94/08012 discloses increasing secretion of over-expressed gene products by co-expression of a chaperone protein such as a heat-shock protein or PDI. Eur. Pat. Appl. No. EP 509,841 discloses increased secretion of human serum albumin from yeast cells using a co-expression system involving PDI and a protein.
The formation of disulfide bonds is essential for the correct folding and stability of numerous eukaryotic proteins of importance to the pharmaceutical and bioprocessing industries. However, numerous studies over the last fifteen years have demonstrated that, with few exceptions, multidisulfide proteins cannot be expressed in active form in bacteria. The production of technologically important proteins with four or more disulfides is costly and complicated and has to rely either on expression of higher eukaryotes that provide a favorable environment for the formation of disulfide bonds or refolding from inclusion bodies (Hockney, 1994; Georgiou and Valax, 1996).
1.2.4 Deficiencies in the Prior Art
Currently there is a lack of efficient methods of producing complex eukaryotic proteins with multiple disulfide bonds on an economic scale. Likewise, there is a need to develop methods which produce proteins that are correctly folded and active without the need for reactivation or subsequent processing once isolated from a host cell.
Therefore, what is lacking in the prior art are methods, recombinant vectors, host cells, and compositions comprising high-level expression of eukaryotic disulfide bond-containing polypeptides (such as trypsin inhibitor, tPA, and variants thereof) which are soluble, correctly-folded, biologically-active, and readily-isolatable from cell extracts of prokaryotic hosts. In particular there is a need to produce tPA and variants thereof which are biologically active, correctly-folded, and localized to the soluble fraction of bacterial cells.